1. Field of the Invention
The present invention relates to a plant in which a phenoloxidase gene is introduced, a method of producing phenoloxidase using the plant, and a method of decomposing and removing hazardous chemical substances using the plant.
2. Description of the Related Art
Phenoloxidase is an enzyme which converts phenols to o-quinone or p-quinone by oxidization, and laccase is a representative example. Laccase is widely present in animals and plants, and fungi, and in recent years laccase could be obtained as an extracellular enzyme of a microbial cell having the ability to produce laccase. Laccase as an extracellular enzyme is produced and purified by purifying a culture filtrate of cells capable of producing the enzyme, or by conducting genetic recombination of laccase genes into hosts e.g. fungi such as Aspergillus genus, culture of the transformant in a liquid medium, and collection of laccase from the culture solution (YAVER, D. S. et al. (1996) Appl. Environ. Microbiol. 62: 834–841, Berka, R. M. et al. (1997) Appl. Environ. Microbiol. 63: 3151–3157). However, in producing laccase using microorganisms capable of producing laccase, particularly the above recombinant, the production efficiency is enhanced, but in the meantime a problem arises that culture and purification etc. are costly. Therefore, although there have been many reports on the usefulness of laccase in wastewater treatment (e.g. coagulative precipitation treatment of organochlorine compounds in pulp bleaching drainage), decomposition of hazardous chemical substances (e.g. decomposition of chlorinated phenols) (Roy-Arcand, L and Archibald, F. S. (1991) Enzyme Microbial Technol. 13: 194–203, Ricotta, A. et al. (1996) Bull Environ. Contam. Toxicol. 57: 560–567, Johannes, C. et al. (1996)Appl. Microbiol Biotechnol. 46: 313–317, Hoff, T. H. O. M. et al. (1985) Appl. Environ. Microbiol. 49: 1040–1045, Chivukula, M. U. R. A. and Renganathhan, V. (1995) Appl. Environ. Microbiol. 61: 4374–4377, Bollag J. -M. et al. (1988) Appl. Environ. Microbiol. 54: 3086–3091, Amitai, G. et al. (1998) FEBS Lett 438: 195–200), production of artificial lacquer paints, turbidity prevention for beverages, clinical analysis, etc., in fact, laccase has not come into practical use. Thus, laccase is brought to a commercial stage only for washing and decoloring denim, which is considered to have high added value.
Accordingly, there is a demand in many fields for a simple production method of phenoloxidase, in particular laccase, which has high production efficiency and desirable cost performance. Though there have been confirmed many patent applications or documents on production methods of phenoloxidase, e.g. laccase (Japanese Patent Application Laid-Open (kokai) No. 9–56378, Japanese Patent Application Laid-Open (kohyo) No. 9–503126, Japanese Patent Application Laid-Open (kohyo) No. 9–505481, YAVER, D. S. et al. (1996) Appl. Environ. Micorbiol. 62: 834–841, Berka, R. M. et al. (1997) Appl. Environ. Microbiol. 63: 3151–3157, etc.), a method which is satisfactory from a practical point of view has not yet been found.
On the other hand, physicochemical treatments are under development as treatment technologies for cases wherein hazardous chemical substances such as PCB, BHC and DDT which are produced as industrial chemical substances, or dioxins which are unintentional products, are stocked at high concentrations or accumulated in an environment. For example, photochemical decomposition, supercritical decomposition, solvent extraction decomposition, catalytic oxidization, vapor phase hydrogenation reduction, melt combustion, heat treatment in reducing atmosphere and glassification treatment are under validation testing. However, these physicochemical methods are not practical, from a viewpoint of cost-effectiveness, for hazardous chemical substances accumulated at low levels in an environment such as in the soil or in rivers, and moreover in situ treatment methods for these substances are required. These hazardous chemical substances diffused over a wide area, even though their concentrations are low, have sufficient concentration levels for endocrine disruption. As a means to overcome this problem, bio-remediation has been conducted with the use of microorganisms that strongly decompose hazardous chemical substances, but such a decontamination method by microorganisms still has drawbacks. That is, the inoculation of the microorganisms and application of nutrient sources needs to be conducted in order to maintain dominance of such microorganisms over a long period, and this becomes more difficult as the contaminated area expands.
From this perspective, in recent years, attempts at decontamination have been made by phyto-remediation (restoration of the environment by plants) which utilizes plants. Plants can be grown independently taking nourishment from the sun, water, and inorganic ions, and can be cultivated extensively by controlling their seeds. Because of this, they have attracted attention as a sustainable environmental decontamination method.
The phyto-remediation that has been examined includes use of detoxification mechanisms or transpiration ability which plants inherently possess. Further, attempts to strengthen the environmental decontamination function of plants have recently made by introducing microorganism-derived genes. However, environmental remediation by transformant plants that has been examined so far involves, in the case of, for example, agricultural chemicals, heavy metals, or the like, transportation and accumulation of these substances to and in cell fractions. Therefore, when the plants die, the accumulated environmental contaminants are released again into the environment and thus this does not lead to a fundamental solution for decontamination. Furthermore, in the case where the hazardous chemical substances are dioxins or PCB, it is predictable that readily degradable substances are decomposed while difficult-to-degrade and highly toxic substances are condensed and accumulated. Thus, there is no other choice to consider that conventional phytoremediation is insufficient.
Against this background, attempts to decompose hazardous chemical substances directly in plant cells using transformant plants into which an enzyme gene for decomposing hazardous chemical substance derived from microorganism are introduced, have been made with respect to 2,4,6-trichlorophenol (Japan Society for Bioscience, Biotechnology, and Agrochemistry, Abstracts for the Annual Meeting, p164, 1998) or γ-hexacyclohexane (Japan Society for Bioscience, Biotechnology, and Agrochemistry, Abstracts for the Annual Meeting, p89, 1997).
Incidentally, it has been clarified that laccase can decompose various chemical substances which are not readily degradable. Laccase can oxidatively decompose endocrine disrupting chemicals including chlorophenols, agricultural chemicals, polycyclic aroma hydrocarbons, alkyl phenol, aroma hydrocarbons, and nitro compounds.
Accordingly, when genes for phenoloxidase, e.g. laccase, are incorporated and plants which can express a function of the genes are prepared, a method of producing phenoloxidase at high yields and desirable cost levels can be established. Also, it is further possible to accomplish phyto-remediation which is useful for decomposing and removing hazardous chemical substances in the environment.
Although there have already been reports (Japanese Patent Application Laid-Open (kokai) Nos. 6-125782, 8-051986, etc.) on methods for obtaining transformant plants by introducing various foreign genes into plants, it is difficult to introduce active phenoloxidase into plants and enable stable secretion and production of the protein locally from roots thereof. Until now there have been no reports on preparation of such transformant plants, methods of producing phenoloxidase by such plants, and phyto-remediation utilizing such plants.